DESCRIPTION (provided by investigator): The discovery of the anti-viral APOBEC3 proteins is regarded as one of the most therapeutically promising breakthroughs in HIV/AIDS molecular virology in recent years. HIV pathogenesis depends on its accessory protein Vif, which functions to counteract the APOBEC3 proteins and trigger their degradation. Five APOBEC3 proteins are susceptible to Vif-mediated degradation, and all seven APOBEC3 proteins have been reported to be capable of individually suppressing the replication of Vif-deficient HIV. Given the additional capacity of these proteins to homo- and hetero-oligomerize, it is further possible that the APOBEC3 proteins form a combinatorial restriction network with a broader range of anti-viral activities exceeding those of individual proteins. Thus, this application has three major aims, each testing an innovative hypothesis that will positively impact the present state of the field. First, we will test the hypothesis that at least two of the understudied APOBEC3s contribute to HIV restriction in vivo. A combination of methods will be used to identify the minimal set of APOBEC3s required for restriction in non-permissive T-cell lines and pathogenesis-relevant primary CD4+ T lymphocytes including gene targeting, short hairpin RNA knock-downs, differentially APOBEC3-susceptible virus isolates, and novel APOBEC3 inhibitor compounds. We anticipate that these studies will enable us to show definitively which APOBEC3s contribute to HIV restriction. Second, we will directly test a combinatorial restriction hypothesis through systematic APOBEC3 co-expression, co- immunoprecipitation, co-encapsidation, single cycle, and spreading infection experiments. Third, we will use APOBEC3-null T-cell lines and DNA deaminase inhibitors as tools to quantify the contribution of the endogenous APOBEC3 proteins to the HIV mutation rate and spectrum. These studies are anticipated to have important implications for understanding virus evolution, immune evasion, and drug resistance. Taken together, this new knowledge will advance our fundamental understanding of the HIV-relevant human APOBEC3 repertoire and, importantly, help prioritize ongoing and future efforts to develop novel HIV/AIDS therapeutics that work by modulating this network. PUBLIC HEALTH RELEVANCE: A detailed molecular definition of the human APOBEC3 proteins relevant to HIV restriction will facilitate the development of novel HIV/AIDS therapeutics that work by leveraging this critical innate immune defense system.